The immobilization of test molecules or “probes” on array supports has had a significant impact on drug discovery, medical diagnostic methods, and basic research. The use of high-density microarrays of organic molecules permits literally thousands of assays to be simultaneously performed on one or more samples. Using high-density microarrays, numerous analytes can be simultaneously detected and/or quantified permitting the rapid characterization of complex systems (e.g. complex assays for gene expression). High-density microarrays are also useful for “high-throughput” screening assays, diagnostics, and in many other contexts. The ability to manufacture microarrays in an efficient and cost-effective manner is of considerable interest to researchers worldwide and of significant commercial value.
In general, microarrays of greater density are preferred. A higher density array typically allows more assays to be performed simultaneously and/or, for lower sample volumes to be used for the same number of assays. In providing large, high-density arrays of molecules (e.g., probes or analytes) there are a number of considerations. The array elements (e.g. dots) should be substantially reproducible in size, particularly if one wishes to quantify an analyte. In addition, the array elements should be consistently and reliably positioned, and should be highly reproducible.
The basic approaches for generating arrays of test molecules such as nucleic acid, protein or other organic molecules fall into two general categories. In the first such approach the test molecules are directly synthesized onto the array support, while in the second such approach the test molecules are attached to the support post-synthetically. Each approach has its own limitations and drawbacks. For example, when an array is created by direct synthesis onto an array support, the efficiency of each synthetic step affects the quality and integrity of molecules forming the array. The magnitude of the problem increases with the complexity of the individual molecules, potentially resulting in an undesirable percentage of incorrectly synthesized molecules and incomplete sequences. Such contaminants can interfere with subsequent use of the array.
In addition, synthetic approaches (e.g. as described by Southern et al. (U.S. Pat. Nos. 5,770,367, 5,700,637, and 5,436,327), Pirrung et al. (U.S. Pat. No. 5,143,854), Fodor et al. (U.S. Pat. Nos. 5,744,305 and 5,800,992), and Winkler et al. (U.S. Pat. No. 5,384,261), are generally unable to construct microarrays of large macromolecules. Such technologies can also be expensive and difficult to implement.
In contrast, the second approach to array production allows the desired molecules to be produced (e.g. synthesized, isolated, amplified, e.g.) by conventional methods prior to their formation into an array. Consequently, the quality of the arrayed molecules, and thus the quality of the resultant array, is potentially greater than that produced by the direct synthesis approach.
Such “spotting” approaches include, but are not limited to inkjet, and direct surface contact printing. Inkjet devices require high reagent volumes and risk “probe” degradation during volatilization.
Direct surface contact printing (see, e.g., U.S. Pat. Nos. 4,981,783, 5,525,464, 5,770,151, and 5,807,522), are limited in their ability to reliably, reproducibly, and uniformly apply the array elements to the array substrate. Reagent usage is also relatively inefficient, and array density is limited.